Cytochrome c oxidase is the multisubunit enzyme complex of the mitochondrial inner membrane which transfers electrons from cytochrome c to molecular oxygen and conserves energy as a proton gradient. Characterization of the human gene for the largest nuclear-encoded subunit (subunit IV) of cytochrome oxidase (COX4) led to the identification of a closely linked expressed gene of unknown function. COX4-associated locus (COX4AL) is linked in a head-to-head arrangement with COX4 and thus the intergenic region (250-bp) likely functions as a bidirectional promoter. The first aim of this project is to determine a possible function of COX4AL by tracking the subcellular location of the 24-kDa encoded protein via immunofluorescence and by determining its association with other cellular proteins using genetic and biochemical approaches. Bidirectional promoters usually encode proteins of similar or related function; thus the specific hypothesis that COX4AL, like COX4, encodes a mitochondrial protein will be tested. A second aim is to ascertain whether the COX4/COX4AL intergenic region functions as a bidirectional promoter. Genetic analysis of transfected promoter-reporter gene constructs in human cells will be used to identify promoter elements essential to the expression of each gene. Third, conserved promoter elements in COX4/COX4AL genes will be used to scan the promoters of other coordinately controlled mammalian genes, including those for other nuclear-encoded cytochrome oxidase subunits and bidirectionally controlled genes, to identify new factors that regulate gene expression or to establish new patterns of control used by well-characterized factors. The long-term objective is to contribute to the functional database of the human genome for a region of chromosome 16 shown to encode the cytochrome oxidase subunit IV gene. Future progress in understanding the molecular bases for human disorders depends on having a complete catalog of the functions of human genes and intergenic regions as a "roadmap" through long stretches of sequence data.